Wound Healing Assay - Science method

Hello Lidia Maeso,

You may perform wound healing assay using either established cell lines or primary cells.

When you perform such assay, it is important that you maintain

conditions as consistent as possible for a successful assay. For established cell lines, culturing protocols which include information such as the recommended growth medium, sub-culturing and expected doubling time are provided to you. But for primary cells, the protocols are often developed by the individual and are often more difficult to standardize.

I prefer using cell lines, though primary cells closely mimic the physiological state of cells in vivo and are expected to maintain their in vivo functions under optimal conditions for a short period of time.

Among cell lines, you could either use fibroblasts or keratinocytes. Both are

the major cell types that respond to the inflammatory phase in the cutaneous repair/regeneration process. Inflammatory signals activate the proliferation and maturation of these two cells types, which is essential for wound healing. Among the fibroblasts, you could use 3T3L1 cells and for keratinocytes you could use HaCaT cell line.

Best.

Dear Dr. Khursheed Ali

You may follow the protocol modified by the investigators in the below attached article. This protocol has been developed as a low-cost handmade method and will prove useful for semi-adherent cell lines. Refer to the protocol given in Figs 1, 2 and 3. The assay is called pipette tip gap closure migration assay especially meant to study cell migration or wound healing in semi-adherent cell lines and may be used as an alternative to the other migration assay protocols.

Regards,

Malcolm Nobre

Hi Sofya Landynya

Yes, The terms "scratch assay" and "wound healing assay" are often used interchangeably, but there are subtle differences between them:

Similarities:

Differences:

In summary:

Please find the protocol attached.

1. https://www.nature.com/articles/nprot.2007.30

2.

Best of luck with your experiments.

Thanks,

Hi Noor Ul Ain,

If you are incubating the cells for shorter time points (less than 12 hrs), keeping the 10%FBS is fine, but for longer time incubation for scratch close, it is recommended to keep them in low serum media. I always incubate cells at least 24 hrs in 0.2% FBS media before introducing the stratch. You can see the figure below from my publication to get a feel on how the growth curves changes with/without Serum.

I'm not sure what you are trying to ask? Media containing 5-15% FBS is frequently used for mammalian cell/tumor cell tissue culture; and Mitomycin C is a highly cytotoxic DNA cross-linking agent, used as an anticancer agent. They serve two entirely different functions, and can't replace each other.

However, co-transfection with MCRA (the gene for mitomycin C resistance) can be used as a selection marker in tissue culture experiments. see the below reference.

Novel selection marker for mammalian cell transfection

Raymond P Baumann , David H Sherman, Alan C Sartorelli

Thank you very much! Your answer helped me a lot

Thank you very much, Andreea Iosageanu and Malcolm Nobre

I had done a pilot experiment previously with 10000 cells per well which gave me nearly 100% confluency. That ought to cover it. Thank you both again

Hello Hilmi Jaufer T a

For longer term wound healing assays (> 24 hr.), you will require mitosis inhibitors such as mitomycin C treatment to eliminate the contribution of cell proliferation to gap closure, allowing the assay to specifically assess migration of the given cell population.

However, in your case, with incubation time of 24 hours and the doubling time of BV2 cell line being around ~35 hours, it may not be necessary to use mitomycin C.

Best.

Dear Nancy,

There are some small cameras for photographing with a microscope, which have a cheap price and are connected to the eyepiece of the microscope.

It is also possible to take photos with the phone camera, which is difficult and may not reach the proper resolution or field of view.

regards

FBS allready contain plenty of not so cell specific growth factors, different kinds of endothelium have different growth requirements, HUVECS are primary cells obtained from vein of somebody-s umbilical cord, short culture only is recomended (7-10 passages?)

Hello,

Firstly, try to make the scratch when the confluency of your plate is around 60-70%. This needs standardization depending on the doubling time of the cell lines.

Secondly, you may use the edge of the lid of the plate you are using for the assay like a scale and make a linear scratch with a 200ul microtip. This will give you an even and reproducible scratch.

Hope this helps

Best regards

ahmed how are you?

Do you happen to have a picture to clarify better? there is a classification regarding the wound and the evolution of healing, but it would help me to answer you. last year i studied something very similar.(I'm not very good at english)

Hi Michael Skiba . i just saw this article. It might be rele to what you are trying to do.

Avoid a non-linear model in case the diagnostics shows you that a linear model meets the regression assumptions. Your "curves" looks linear, so y_i = b_0 x_i b_1 +e_i (= including the factor variable for the different groups) is one candidate of a linear model, and y_i = b_0 + b_1 x_1 + b_1 x^2_i b_2 + e_i could be an alternative linear model to check.

From the curves shown, I'm in doubt that a non-linear model was fitted, but just the points are connected.

I think you can further clarify the question.

Yes, I have done scratch wound healing on a 96-well plate. I used a 10 ul tip instead of a 100 ul one. also, neither I used a ruler nor lines drawn at the back. however, I used to keep 5-6 wells for each sample. then I used to take images. from each well I was able to take at least 5-6 images. though the scratches were non-uniform, in most of the area thickness of the wound was the same, so out of many images taken, I considered images with the same thickness for the analysis.

I haven't used Hs27 cells before, but 3T3 fibroblasts work well in wound healing assays as you can see in these video's:

3T3 scratch assay (wound healing assay) time lapse - CytoSMART Lux2 - YouTube

The effect of Blebbistatin and Cytochalasin-D on collective cell migration - YouTube

Both are very different assays. Depends on what you are looking into.

If you intend to investigate the effects of cell-matrix, cell-cell interactions etc. you could use the wound healing assay. It's a 2D cell migration approach to semi-quantitatively measure cell migration of a sheet of cells, especially suitable for endothelial cells. in the wound healing assay, you cannot exclude the fact that the cells fulfill the wound simply by proliferation. Also if the scratching done manually there might be some inconsistencies with the depth, size and borders of the scratch.

A cell culture insert/transwll/boyden chamber/filter membrane migration/cellqart/chemotaxis assay cell are placed on top of a microporous membrane. After some time only the cells that have migrated through the membrane are stained and enumerated. Particulary useful to investigate the effect of chemokines, cytokines or other soluble substances added to the system or secreted by the another cell type that may or may not influence the migration of your cells of interest.

You can find some additional info in this article:

Dear Mark!

You look at following articles, please:

Thanks for the answer.

We are working on 6 wells plates, so we have an higher density of cells, that's why we are looking for something a little more wide. But i'll follow you advice on applying more pressure

Hello, Evra!

You see this article, please, there is a morphology of PC3 cells

The images after 16h look fine to me. The thread like structures could be because of migration of the cells into the wound. However, the cells density is higher after 16h indicating that the cells are overcrowding. For migration assay, you should use mitomycin C to suppress cell proliferation to make sure that the wound healing is because of migration and not the cell proliferation.

If you must use phase contrast, try larger wells (6 well plates). with the scratch in the center of a 35 mm well, you can avoid strange optical effects from the edge of the well. Still adjust the condenser and phase rings for imaging each well to optimize captured image quality. Also make sure to have enough medium, and the same amoint of medium in each well to avoid meniscus effects.

Hi All,

I just realised that I have completely forgotten to reply for almost a year! I did manage to optimize the protocol, and in the end my HUVEC got a pretty good response to VEGF (more than 150% migration 18 hours after treatment). I think the trick here is not to add BBE or any other supplements into the EBM starve media, other than FBS (0.5%), Glutamine and ascorbic acid. It is possible that these other supplements/growth factors may have other effects on the HUVECs (e.g proliferate) rather than migrate, which may be why I didn't see a response to VEGF. Also I did starvation for 4-6 hours only and not overnight.

Thanks for all your contributions! If you have an even better protocol, please do share.

Management of Keloid Scars: Surgical Versus Medical Therapy

A BrdU incorporation assay, DiviTum, can be used in cell cultures, mouse and human sera/plasma. Dr Klaus Felix from Heidelberg have published results using DiviTum in pancreas PDAC:

Hi Shehnaz Ahmed ,

I have been trying to find an answer to your orignial question, with no luck. Did you happen to find a solution?

Kind Regards,

Nick

Which kind of DMEN are you using?

I was trial different types of DMEN, and the best is Gibco™ DMEM, high glucose, GlutaMAX (#11574516).

Hi i would like to ask you if has already happened to you that when the wound is made in the hacat cells if it opened mainly in the middle of the well. I tried to take off all the medium DMEM with 10% SFB in order to the wound do not open but this does not resolve the problem. I would really appreciate if someone could help me. Thanks!!!

It depends on what type of media you use. I use an endothelial cell media, you can either use EGM media from Lonza or ECM media from ScienCell. From my experiments, the EGM media allowed rapid growth but the ECM media allowed constant maintenance of the cells.

To avoid that, we pretreated plates with fibronectin and then seed cells onto fibronectin-treated wells.

I found an answer to the question. Dr. Ce´dric Blanpain lab applied 5-fluorouracil (5-FU) to block epidermal cell proliferation. Please refer to: DOI: 10.1038/ncomms14684

,

Hello Marian.

Better to check both ways to find the best working of your assay system.

Trial 1> Vehicle alone [(-)ve control], Arab-C addition for 48h (treated), Arab-C addition + protein (healing) and Vehicle alone + protein [healing on (-) control] groups: Arab-C and protein are added once.

Trial 2> Arab-C addition for 48h (treated), then release from Arab-C by complete washing gently. (-)Protein (healing) and (+)protein groups.

You could set up 24h, too, depending on cell viability test first.

Best wishes.

If you feel that any answer is helpful to you, then recommend such answer/s.

L929 (fibroblast) and HaCaT (keratinocyte) cell lines for scratch assay. What is the cost of this assays at APT ?

1. Serum has VEGF.

2. Heparin binds to too many stuff in addition to VEGF (eg. HB-EGF)

3. VEGF-A Neutralizing antibodies are the best.

Extract only works on some cells, but not all. Just like some cytotoxicity assay, some of the plant extract can suppress the growth of certain cell lines, but not all, because the mechanism inside the cells are different. 

Or you can try not using FBS when u want to incubate the cells with the extract, starvation allows the cells uptake more extracts. :) 

Hi Devrim, I assume you are doing a wound healing assay using tissue culture cells, not in vivo models.

If you are looking for inhibition of migration across the scratch in the plate, you have to use sub-confluent cells (~50% confluent). One plate with cells without inhibitor and one plate with inhibitor.

Personally, I tried the scratch assay and found that it is very difficult to do consistently. If the scratch is too deep, then cells physically cant cross it. If the scratch is too shallow then you have problems seeing the line.

I gave up on it and used transwells.   

did you coat the wells with gelatin for migration( woung healing) and with matrigel for tube formation?

 Thank you very much for your help. Finally it has already solved with better aseptic conditions.

Before setting up experiments, you should first concentrate on the markers you want to study depending on the field you are working in and the type of cancer. There are indeed different markers for cancer stem cells depending on the type of cancer. Pay also attention on the field your are studying EMT, is it after radiotherapy or during any other treatment... Then you can certainly make immunoblot or flow cytometry depending on your conditions and your markers, but also migration test and sphere formation for a mechanistic approach

Hi Elisa,

Where did you get stuck? Assuming that you are following the steps described in the documentation, you need to find the right parameters for your images:

You may also find this thread interesting:

Here is an alternative method:

All the best,

Eszter

Hi Aurélie, You need to add Cytosine β-d-arabinofuranoside hydrochloride (Sigma) (10 μM), a selective inhibitor of DNA synthesis, which does not inhibit RNA synthesis. 

You can find a protocol in this paper: https://www.ncbi.nlm.nih.gov/pubmed/24904530. Good luck

I agree with Dr. Mitchell,

Formulate it in cream or gel or ointment. I think dont wrap or close wound after application and also no need to remove the eschar. Apply the drug at sides of the borders of eschar and also on the Top. If you dont want to make formulation then you can add your solution drop by drop if want specific quantity. If quantity does not matter then add with completly soake sterile cotton.

Dear Vincent,

you can see a detailed description of how to interpret the results in our website, in the following link:

Thanks for your interest

We have made hundreds of stably expressing cell lines.  If you have two different genes you wish to express that are currently in two different plasmids, I would  linearize them ,  transfect them together, and select for your resistance marker (neo or puro or whatever).  Cells that integrate the plasmids will often integrate both plasmids, usually with multiple copies.   Once you start getting colonies and you clone these out, you can test your individual lines for expression of the transgenes.  If you screen enough of them, you will find some that express both proteins inserted.  Of course, all of this assumes that expression of your transgenes is not lethal or inhibitory to cell growth.   Expect to screen at least 20-50 cloned lines.  

If you are looking for a +control that speeds migration, you can add TGFbeta1 to the media right after wounding. This is both well documented in literature and physiologic since this cytokine is abundant in wounds and causes activation of fibroblasts (to myofibroblasts). 

Dear Ramin,

the solubility of the allantoin in the water is 0.57 g/100mL at the 25°C, therefore it is not possible to prepare 5% stock solution in the water and the test concentration is just on the solubility limit. Try solving the allantoin in the alkaline buffer, but I do not know if the alkaline pH is compatible with your experiment.

Best regards

Pavel

thank you for your reply I will try the scratch assay and see if cell can migrate in the scratched area even with the coating is damaged 

For initial assessment of the potential of your drugs first in vivo wound healing model must be mice model of incisional wounds. This model minimizes artifacts and reveals full potential of your drugs on different wound healing stages and processes.  The study should be conducted on healthy mice in order to examine in parallel both efficacy and safety of the drug candidates. From my personal experience (which is development of several dermal/wound healing drug candidates from basic research to advanced stages of clinical development) I recommend to pick up only those leads that are able to improve wound healing in healthy mice.   

Article that describes the model is –  “Novel insights into wound healing sequence of events.”

Abstract - http://www.ncbi.nlm.nih.gov/pubmed/17943650

Full text - http://tpx.sagepub.com/content/35/6/767.long

I may address any questions you have regarding the article and the model. Moreover, since that article we improved the assessment protocol and increased clinical relevance of the model. This work still not published, but I may share with you some details if needed. Good luck!

If you still have the plates, you could try staining a well with Ponceau S dye. If you first coated with gelatin, the scratch will be red.

If your cells are of good quality no coating is needed, you just have to work with good cell density (low cell density causes stress). When you perform scratches coating agent is removed, at least partially. This will cause variability and artifacts. Moreover, these cells behave differently on different types of coating – meaning that coating itself may cause artifacts related to cell migration.

I recommend you to start from relatedly high cell number – seed such amount that will give you sub confluent plates within 24 h. After that do scratches and make pictures for analysis every 24h (do not forget time zero before treatment, just after you did scratches). For proper analysis of migratory capacity you should prepare two sets of replicates – one in regular conditions and another with inhibited proliferation. For inhibition of cell division you may use Mitomycin C which is commonly used to generate mitotically inactive cells in culture.

With regard to scratches manual, in each plate we do two parallel horizontal lines and two parallel vertical lines. This gives you from each plate 12 roads and 4 junctions to take pictures from. Try to mark areas (on the bottom) where you take pictures, and every day take the same frames. In such a way, even if you work with triplicates only, you will have enough pictures for digital measuring and for proper statistical analysis (Anova, t-test and Cohen's d).

Good luck!

Dear Saleh Alkarim,

Thank you for your opinion!

Also appreciate for your links.

Hello Lee Jinkyu.

I don´t quite understand your question. Are your pictures affected by the flash? Do you see a big white circle, or are they somehow not evenly illuminated? If this is so, of course this affects the areas calculated. Most of these programs are based on threshold and pixel-counting. If you have a big white area on your pictures, then this might have an effect on the calculated area.

If you are wondering whether the flash per se has an effect on your cultures: I don´t think so. Although it has been shown that irradiation with 635 nm (a part of visible light) reduces intracellular ROS production in HUVECs, which results in increased angiogenesis (see publication below):

Greetings

In my opinion standard antibiotics (like penicillin/streptomycin) are there only to prevent bacterial growth/contamination. Most likely they don't have much impact on the result in this assay. However, it's obviously best to be consistent with the protocol to get comparable results - either always use antibiotics or not. So, in my opinion antibiotics are not required.

Personally I have routinely used penicillin/streptomycin when culturing HUVECs and also used strictly sterile work procedures. Antibiotics won't prevent all microbial contamination and for example mycoplasma can go unnoticed unless specifically tested.

Vaseline (petrolatum), however, can delay epithelial migration, so any improvement you see in your extract may be due to cell mobility more than any active property. I would think that you would need some kind of cover (gauze) for both groups, so containing a hydrogel wouldn't be a problem. Hydrogels aren't very watery, actually. They're more like lubricating jelly (eg: KY) than a liquid.

Hi

I found this

seems the salution!

good luck

Carlo

Dear Kirsten,

Please read the following:

Cytometry A. 2004 Sep;61(1):35-44.

Effects of fluorescent and nonfluorescent tracing methods on lymphocyte migration in vivo.

Nolte MA1, Kraal G, Mebius RE.

Author information

 

Abstract

BACKGROUND:

The use of fluorescent dyes to monitor in vivo cellular migration and proliferation has greatly expanded, but little is known about their potential influence on cell migration.

METHODS:

Adoptive transfer studies of lymphocytes labeled with various dyes were performed, and their in vivo homing was compared with that of coinjected unlabeled control cells. In addition, in vitro migration and binding studies were performed to analyze the various steps of transmigration separately.

RESULTS:

These data showed that the intracellular fluorescent dyes calcein acetoxymethyl ester, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, and fluorescein isothiocyanate affect in vivo homing of especially B lymphocytes to lymphoid organs, without any direct effect on in vitro chemotactic or adhesive activity. The only label that did not affect migration was the extracellular and nonfluorescent molecule biotin, provided that the labeling was performed at room temperature. Interestingly, by using the highly versatile congenic Ly5.1-Ly5.2 system, we also demonstrated intrinsic differences in lymphocyte migration based on allelic differences.

CONCLUSIONS:

Our data showed that fluorescent labeling of lymphocytes has a severe effect on their homing capacity in vivo. Labeling of cells with biotin appeared to be a good alternative for this purpose; however, if direct fluorescence is required, the negative effects on cell migration should be considered.

Hoping this will be helpful,

Rafik

I do not hav any experience with imagej Software. But the main use of ImageJ is that it can calculate area (the area will reduce gradually -serial analysis required) and pixel values which may be useful for graphic designers.

Wound color composition and texture may be analysed by wound imaging (pixel color). It may be possible to diagnose healthy and unhealthy wound bed. Please read that may help you to solve your problem- Nayak R, Kumar P, Galigekere RR. Computer assessment of the composition of a generic wound by image processing. Plast Aesthet Res 2015;2:261-5

Dear Prabhakaran Selvam

The article is: "Review of Natural Compounds for Potential Skin Cancer Treatment". Best regards

Dear Lee,

Generally, the plates are  coated  with Gelatin 0,2% and sometimes 1% for about 30 minutes to 40 minutes.

The following paper discusses coating by gelatin:

Biotech Histochem. 1992 Jul;67(4):241-50.

A comparison of substrates for human umbilical vein endothelial cell culture.

Smeets EF1, von Asmuth EJ, van der Linden CJ, Leeuwenberg JF, Buurman WA.

Author information

 

Abstract

We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.

Specific procedure for coating by gelatin:

The procedure .

1. Prepare 1mg/ml gelatin stock solution, dissolve by incubating in 37 degree water bath, filter it (to remove any floating materials) and finally autoclave it.

2.In the day of the experiment, coat the T-25 flask with aprox. 2ml of 1mg/ml gelatin (it should cover the surface of the flask) in side the hood and keep it for 1hr inside the hood.

3. After 1hr remove excess gelatin solution and use the flask for tissue culture.

The following is a protocol with collagen:

• Culture medium

M199 “stock”:

- M199 Earle 10X 100 ml

- Distilled water 900 ml

M199 “full”:

- Glutamine 0.2 M 1 ml

- HEPES 1 M 1.5 ml

- NaHCO3 7.5 % (w/v) 1.8 ml

- Penicilline/streptomycine (10000/10000) 1 ml

- FCS (heated at 56°C and centrifuged) 20 ml

- M199 “stock” up to 100 ml

Conservation at +4°C up to 1 week.

II- Cell culture

• The day before the manipulation:

- Coat the Petri dishes (35 mm; 7 dishes/cord) with 1 drop of fibronectin

- Bring at the maternity 3 recipients containing 50 ml of conservation buffer for cords

• The day of the manipulation:

- Fill up a recipient with ~ 500 ml of physiologic serum and maintain at 37 °C (“bain-marie”)

- Under the hood, prepare (for 1 cord):

¾ 2 syringes of 50 ml, 1 syringe of 30 ml and 1 syringe of 10 ml

¾ 1 surgical clamping clip, 2 cannulas, string, sterile compresses, sterile scalpels and

sterile gloves

- Spread on the working area under the hood: 1 aluminium foil, 1 paper sheet and the

“sterile envelope” of gloves (in this order)

3

• Cord manipulation and culture:

- Tidily cut the 2 ends of the cord with scalpel

- Introduce a cannula at each extremity of the vein (the widest vessel) and tightly maintain

it with string

- Wash the cord with PBS using the syringe of 50 ml

- Inject the collagenase (0.2 %) at one end of the vein using the syringe of 30 ml

- When leaking out the other end, tightly clamp it with the surgical clip

- Maintain the syringe and protect the cord’s ends with “clean” aluminium foil

- Incubate the cord 10 min in the physiologic serum at 37 °C

- Upon the sterile envelope of gloves, gently squeeze the cord

- Fill up a sterile falcon of 50 ml with 10 ml of “full” culture medium

- Collect the cells in this falcon by washing the vein with 40 ml of PBS

- Centrifuge at 900 rpm for 10 min

- Carefully discard the supernatant and suspend cells in ~14 ml of “full” culture medium

- Dissociate the cells (aspiration and repulsing X 3) with the syringe of 30 ml with needle

- Add 2 ml of the cell suspension in each fibronectin-coated Petri dish

Cultures are placed in a humidified 95% air and 5% CO2 atmosphere at 37° C.

The following day, non-adherent cells are removed by changing the culture medium. The culture medium is changed every two days and confluency is typically achieved in 6-8 days with “cobblestone appearance” in optical microscopy (~ 1.10 6

cells/dish).

Hoping this will be helpful,

In my opinion you should focus on the genes/proteins associated with EMT (Epithelial to mesenchymal activation). To start with, check the protein levels for EMT markers like as Damiano said E cadherin (it goes down in the cells acquired the migratory behaviour i.e acquiring mesenchymal traits),Vimentin(goes up, highly studied protein in migratory and invasive behaviour), N-Cadherin (reported to goes up in EMT), cell junction proteins (this may go down as well, as cell loosing its epithelial phenotype and acquiring the migratory behaviour...so these may include checking protein expression from Desmoplakin 1/2, plakoglobins etc).....Thats it!...sufficient to start with for the welcoming year!

Hi Abdul, 

Invasion: what do you mean you didn't observe any difference in 24hrs? Do you mean no cells have invaded? In this case how did you assay for this? Staining the underside of the membrane or simply visually? Did you rehydrate the membrane with media before adding matrigel? The assay may require optimisation with some very invasive cells (e.g. MDA231), I'm not sure how invasive BT474s are. 

Migration: In my opinion 7 billion cells is way too high a seeding density. Ideally you want to let the cells divide a few times until they reach confluence and then scratch. Seed 0.3-1e06 cells then let them reach confluence over a couple of days. If you scratch an over-confluent monolayer then you will get the "flap" of cells every time so I would definitely avoid over seeding.

If you have to use a tip to inflict the wound then be sure to scratch only once with a p10 (p200 is too wide). If you have access to an esson wound maker I would recommend using this. If not consider using an insert (link) to get a uniform scratch area. One of the most important things in a wound healing assay is to ensure wounds are uniform across replicates and that becomes very difficult when you are applying the wound by hand. 

144 days is far too long to assay migration, in this time frame you will have to contend with cells dividing in the wound itself, not migrating from the edges. You should be looking at 12-36 hours maximum in low serum media if you want to assay cell migration and not cell division. 

Hope some of this helps! 

John 

By the way Manuka honey is not the only one available and as you can see in the small trial we did in the wound clinic Revamil ( Holland ) proved itself very efficient and better than sulfadiazine stengthening the opinions of Dr Traynor and Dr Lambrecht and other respected autors.

To mention that not every honey has the same characteristics and efficiency.For instance Manuka honey has glyoxal and Revamil has Bee Defensin.The concentration,the dressing'the ingredients mixed with the honey ( gel- balm...) have all their advantages and inconvenients but maybee it is already the luxury of "developped "countries  and Dr Lambrecht with our collegues of Asia that published a lot of works on honey treatments for wounds and burns would turn it more simple.

For myself I can say that I got the respect of my family when I made them discover the Revamil balm for superficial burns ....

kindly yours

The means of wound area measurements between groups at different time intervals can be compared using one-way ANOVA followed by Tukey’s post-hoc test.

To examine the mean difference in epithelization can be one-way ANOVA.

To examine the mean differences in wound healing between the groups in incision and dead-space wound models can be one-way ANOVA.

You can measure the degree for blood clotting in the lab when you add this plant extract on blood cells

I have in the past done castings of cavity wounds. I used dental impression molding material. You can analyse dimensions from that perspective. 

I also though, relied more heavily on monitoring wound healing progress using the Wound Assessment Parameter Scoring Tool (WAPST) 

Kathi

I use a lot of the dressing Acticoat in my practice, and have studied it extensively in clinical trials and in vitriol. Acticoat contains nano-crystalline silver. I am reliably informed that when water is applied to the dressing the nano-crystalline structure gives off silver ions and silver atoms. The silver ions kill bacteria, and the atoms have an anti-inflammatory action. This is certainly the effect that is seen clinically. Unfortunately the silver also is toxic to keratinocytes. Therefore it is a balance between killing bugs and killing keratinocytes.  For example, Acticoat will delay wound healing if used on non infected burns, but is a superb dressing if used on an infected or dirty wound that has a strong potential for infection.

African green monkey kidney (Vero) cells. Easily establish confluent monolayers and don't require special culture media. Also, Vero cells are very adherent but not super sticky making a multitude of experimental assays easily performed like flow cytometry, immunoblotting, and ELISA (to name just a few). Easily found on ATCC website too.

Semi-quantitative swab cultures are not the gold standard, quantitative tissue cultures are.  If you do need to do a swab culture, then the Levine technique is recommended as the best swab technique.  

Levine, N. S., et al. (1976). "The Quantitative Swab Culture and Smear: A Quick, Simple Method for Determining the Number of Viable Aerobic Bacteria on Open Wounds." The Journal of Trauma 16(2): 89-95.

Hi Sadie,

from your questions is not clear what scientific question you want to answer. What cancer cell you want to compare with HaCaT? SCC? Melanomas? or others? HaCaT have a p53 mutation and come from keratinocytes. Compare HaCaT cells with SCCs or with HaCaT derived SCCs (IL4 cells, A5RT1)! Read Boucamp et al. (around 1990). To answer your question: yes, you can possibly see contradicting results when you compare different cell lines. That would not be surprising. Transwell migration assay is also fine for another assay as suggested by Yumi!

All the best und much success!

Best regards,

Peter

That depends upon your coating technique. I've used 200ug/ml for coating by adsorption to the surface, just in the fridge over night or at 37 degrees for 2-4 hours, but it's a crude method, as you're not really sure how much collagen sticks down.

If you want to know more accurately how much collagen is actually present then allowing a collagen solution of known concentration to dry onto the slide will work, leaving you with an easily calculable value for moles or grams per cm2. For this method I'd suggest much lower concentrations, more in the area of 10ug/ml, but again this depends upon the volume you use as the concentration becomes irrelevant after drying, and the total mass of collagen is deposited upon the surface.

I do this by spreading the collagen solution over the slide with a pipette tip so that it forms a complete covering held under surface tension, and then leaving it in the flow hood overnight to dry.

In either case, i'd suggest as in initial experiment testing a range of volumes/concentrations to see what works best for your cells and your method. I know it's one more thing to do, but in my experience a simple optimisation step like this almost always saves a lot of time in the long run.

Sorry if this is a little long winded!

Good Luck.

hello

normally for wound healing assay u need  the wound created to be quiet similar (not necessary the same). the distance between the wound influences the rate of closure. yet still u can analyse your result by following this software called tscratch, which automatically does the calculation for you. it uses the initial wound created and wound closure during the expt to give you a measurement of how your treatment varies from the control. please find attached the protocol. for the pipette tip some use 10 ul and others use 200 ul  or even 1ml tips. but this depends on how fast ur cells migrate to wounded area. eg is leukocyte cells they migrate very fast so when u use 10 ul tip, the wound might be close quiet fast. 

cheers sis!!!!!!!!!!!

Dear Rachel,

Vheck this out:

Eur J Pharm Biopharm. 2014 Feb;86(2):277-83. doi: 10.1016/j.ejpb.2013.10.003. Epub 2013 Oct 15.

Wound healing potential of a dimeric InlB variant analyzed by in vitro experiments on re-epithelialization of human skin models.

Kolditz F, Krausze J, Heinz DW, Niemann HH, Müller-Goymann CC.

Abstract

A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.

Right I would agree with both.

Sorry to bring in another twist, but one should be a little bit more careful with the word "wound healing assay. Actually when cell cultures are manipulated such as with scratching, this is not a wound healing, but rather the capability of cells - such as for instance keratinocytes - to overgrow the gap and close the space  "Wound helaing" is by far a much more complex phenomenon that unfortunately cannot be mimicked by a sample scratch assay..

I think I understand my confusion now. In Reviews about wound healing (without an explicit restriction to human or mice) contraction is an integral part (e.g. http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pmc/articles/PMC2736861/ ). By contrast many wound healing experiment papers are mainly concerned with the process of "wound closure", in which in mice, when left alone, the panniculosus carnosus layer plays an important role. Additionally, since mice are loose skinned, "normal" contraction via myofibroblasts leads to greater tissue movements than in humans.

So when papers mention that wound contraction is not important for wound healing in humans, they rather refer to wound closure. For the establishment of "normal", tension-resistant scar tissue, contraction is important in both mice and humans (and if gone wrong leads to fibrosis/hypertrophic scars etc.)

Does that sound about right?